We have previously used cryo-electron tomography combined with missing-wedge corrected, sub-volume averaging and classification to obtain 3D structures of macromolecular assemblies in cases where a single dominant species was present, and applied these methods to the analysis of a variety of trimeric HIV-1 and SIV envelope glycoproteins (Env). Here, we extend these studies by presenting a method for determining the distribution of conformational states found in a specimen and validate these procedures by successfully separating and reconstructing distinct 3D structures for unliganded and antibody-liganded as well as open and closed conformations of Env present simultaneously in mixtures. We show that identifying and removing spikes with the lowest signal-to-noise ratios improves the overall accuracy of alignment between individual Env sub-volumes, and that alignment accuracy, in turn, determines the success of image classification in assessing conformational heterogeneity in heterogeneous mixtures. This development extends the sub-tomogram averaging capabilities to heterogeneous samples. Furthermore, it turns cryo-electron tomography into a powerful analytical tool that can directly determine the relative amount of the different conformations found in the specimen.View Large Image | View Hi-Res Image | Download PowerPoint Slide